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- PDB-9aya: Crystal structure of CRAF/MEK complex with NST-628 and active RAF... -

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Basic information

Entry
Database: PDB / ID: 9aya
TitleCrystal structure of CRAF/MEK complex with NST-628 and active RAF dimer
Components
  • Dual specificity mitogen-activated protein kinase kinase 1
  • RAF proto-oncogene serine/threonine-protein kinase
KeywordsTRANSFERASE / Inhibitor / complex / SIGNALING PROTEIN
Function / homology
Function and homology information


death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / intermediate filament cytoskeleton organization / JUN kinase kinase activity / regulation of axon regeneration / placenta blood vessel development / mitogen-activated protein kinase kinase / MAP-kinase scaffold activity / labyrinthine layer development ...death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / intermediate filament cytoskeleton organization / JUN kinase kinase activity / regulation of axon regeneration / placenta blood vessel development / mitogen-activated protein kinase kinase / MAP-kinase scaffold activity / labyrinthine layer development / cerebellar cortex formation / type B pancreatic cell proliferation / regulation of Rho protein signal transduction / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / Signaling by MAP2K mutants / regulation of cell motility / positive regulation of axonogenesis / insulin secretion involved in cellular response to glucose stimulus / regulation of Golgi inheritance / Negative feedback regulation of MAPK pathway / trachea formation / regulation of early endosome to late endosome transport / GP1b-IX-V activation signalling / regulation of stress-activated MAPK cascade / Frs2-mediated activation / IFNG signaling activates MAPKs / MAPK3 (ERK1) activation / ERBB2-ERBB3 signaling pathway / endodermal cell differentiation / face development / MAP kinase kinase activity / pseudopodium / neurotrophin TRK receptor signaling pathway / regulation of cell differentiation / somatic stem cell population maintenance / Bergmann glial cell differentiation / Uptake and function of anthrax toxins / thyroid gland development / protein kinase activator activity / MAP kinase kinase kinase activity / extrinsic apoptotic signaling pathway via death domain receptors / type II interferon-mediated signaling pathway / negative regulation of protein-containing complex assembly / Schwann cell development / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / activation of adenylate cyclase activity / keratinocyte differentiation / : / response to muscle stretch / myelination / ERK1 and ERK2 cascade / CD209 (DC-SIGN) signaling / protein serine/threonine/tyrosine kinase activity / insulin-like growth factor receptor signaling pathway / protein serine/threonine kinase activator activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / thymus development / Signal transduction by L1 / positive regulation of protein serine/threonine kinase activity / cell motility / RAF activation / wound healing / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / positive regulation of peptidyl-serine phosphorylation / neuron differentiation / Stimuli-sensing channels / Negative regulation of MAPK pathway / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / chemotaxis / cellular senescence / MAPK cascade / Signaling by BRAF and RAF1 fusions / late endosome / insulin receptor signaling pathway / heart development / scaffold protein binding / regulation of apoptotic process / histone H3Y41 kinase activity / histone H2AXY142 kinase activity / mitochondrial outer membrane / eukaryotic translation initiation factor 2alpha kinase activity / positive regulation of MAPK cascade / positive regulation of ERK1 and ERK2 cascade / 3-phosphoinositide-dependent protein kinase activity / DNA-dependent protein kinase activity / ribosomal protein S6 kinase activity / histone H3S10 kinase activity / histone H2AXS139 kinase activity / histone H3S28 kinase activity / histone H4S1 kinase activity / histone H2BS14 kinase activity / histone H3T3 kinase activity / histone H2AS121 kinase activity / Rho-dependent protein serine/threonine kinase activity
Similarity search - Function
: / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / : / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. ...: / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / : / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / C1-like domain superfamily / Ubiquitin-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
: / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / RAF proto-oncogene serine/threonine-protein kinase / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.59 Å
AuthorsQuade, B. / Huang, X.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cancer Discov / Year: 2024
Title: The Pan-RAF-MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS-MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers.
Authors: Meagan B Ryan / Bradley Quade / Natasha Schenk / Zhong Fang / Marshall Zingg / Steven E Cohen / Brooke M Swalm / Chun Li / Ayşegül Özen / Chaoyang Ye / Maria Stella Ritorto / Xin Huang / ...Authors: Meagan B Ryan / Bradley Quade / Natasha Schenk / Zhong Fang / Marshall Zingg / Steven E Cohen / Brooke M Swalm / Chun Li / Ayşegül Özen / Chaoyang Ye / Maria Stella Ritorto / Xin Huang / Arvin C Dar / Yongxin Han / Klaus P Hoeflich / Michael Hale / Margit Hagel /
Abstract: Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the ...Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the phosphorylation and activation of MEK by RAF, overcoming the limitations of traditional RAS-MAPK inhibitors and leading to deep durable inhibition of the pathway. Cellular, biochemical, and structural analyses of RAF-MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF-CRAF heterodimers, a differentiated mechanism from all current RAF inhibitors. With a potent and durable inhibition of the RAF-MEK signaling complex as well as high intrinsic permeability into the brain, NST-628 demonstrates broad efficacy in cellular and patient-derived tumor models harboring diverse MAPK pathway alterations, including orthotopic intracranial models. Given its functional and pharmacokinetic mechanisms that are differentiated from previous therapies, NST-628 is positioned to make an impact clinically in areas of unmet patient need. Significance: This study introduces NST-628, a molecular glue having differentiated mechanism and drug-like properties. NST-628 treatment leads to broad efficacy with high tolerability and central nervous system activity across multiple RAS- and RAF-driven tumor models. NST-628 has the potential to provide transformative clinical benefits as both monotherapy and vertical combination anchor.
History
DepositionMar 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RAF proto-oncogene serine/threonine-protein kinase
B: Dual specificity mitogen-activated protein kinase kinase 1
C: RAF proto-oncogene serine/threonine-protein kinase
D: Dual specificity mitogen-activated protein kinase kinase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,07710
Polymers133,0394
Non-polymers2,0386
Water88349
1
A: RAF proto-oncogene serine/threonine-protein kinase
B: Dual specificity mitogen-activated protein kinase kinase 1
hetero molecules

C: RAF proto-oncogene serine/threonine-protein kinase
D: Dual specificity mitogen-activated protein kinase kinase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,07710
Polymers133,0394
Non-polymers2,0386
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_555x+1/2,y+1/2,z1
Unit cell
Length a, b, c (Å)149.725, 58.202, 162.347
Angle α, β, γ (deg.)90.000, 95.590, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z

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Components

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Protein , 2 types, 4 molecules ACBD

#1: Protein RAF proto-oncogene serine/threonine-protein kinase / Proto-oncogene c-RAF / cRaf / Raf-1


Mass: 31975.775 Da / Num. of mol.: 2 / Mutation: Y340D Y341D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAF1, RAF / Production host: Homo sapiens (human)
References: UniProt: P04049, non-specific serine/threonine protein kinase
#2: Protein Dual specificity mitogen-activated protein kinase kinase 1 / MAP kinase kinase 1 / MAPKK 1 / MKK1 / ERK activator kinase 1 / MAPK/ERK kinase 1 / MEK 1


Mass: 34543.836 Da / Num. of mol.: 2 / Mutation: S218A S222A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAP2K1, MEK1, PRKMK1 / Production host: Homo sapiens (human)
References: UniProt: Q02750, mitogen-activated protein kinase kinase

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Non-polymers , 4 types, 55 molecules

#3: Chemical ChemComp-A1AHE / N-[3-fluoro-4-({7-[(3-fluoropyridin-2-yl)oxy]-4-methyl-2-oxo-2H-1-benzopyran-3-yl}methyl)pyridin-2-yl]-N'-methylsulfuric diamide


Mass: 488.464 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C22H18F2N4O5S / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 49 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.65 Å3/Da / Density % sol: 53.51 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 0.2M magnesium chloride, 0.1M tris pH 7.0, 10% w/v PEG 8000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL02U1 / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER2 S 9M / Detector: PIXEL / Date: May 5, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 2.59→161.57 Å / Num. obs: 43777 / % possible obs: 99.9 % / Redundancy: 4.5 % / Biso Wilson estimate: 49.21 Å2 / Rmerge(I) obs: 0.075 / Net I/σ(I): 13
Reflection shellResolution: 2.59→2.69 Å / Rmerge(I) obs: 0.544 / Num. unique obs: 2754

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
REFMAC5refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.59→57.64 Å / SU ML: 0.3592 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 26.9142
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2566 2115 4.83 %
Rwork0.212 41638 -
obs0.2141 43753 99.83 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 56.57 Å2
Refinement stepCycle: LAST / Resolution: 2.59→57.64 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8765 0 132 49 8946
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00389097
X-RAY DIFFRACTIONf_angle_d0.565212322
X-RAY DIFFRACTIONf_chiral_restr0.04411369
X-RAY DIFFRACTIONf_plane_restr0.00461562
X-RAY DIFFRACTIONf_dihedral_angle_d15.40923328
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.59-2.650.36051290.30732754X-RAY DIFFRACTION99.86
2.65-2.720.31651350.27982718X-RAY DIFFRACTION99.93
2.72-2.790.33571420.25662766X-RAY DIFFRACTION99.76
2.79-2.870.2961320.24852798X-RAY DIFFRACTION99.9
2.87-2.960.3111470.25732717X-RAY DIFFRACTION99.86
2.96-3.070.31851390.27032766X-RAY DIFFRACTION99.93
3.07-3.190.3671290.27172787X-RAY DIFFRACTION99.93
3.19-3.340.31341400.24972750X-RAY DIFFRACTION99.93
3.34-3.520.30531580.23082745X-RAY DIFFRACTION99.79
3.52-3.740.23661410.21172761X-RAY DIFFRACTION99.9
3.74-4.020.24891680.20022769X-RAY DIFFRACTION99.97
4.02-4.430.22541460.16922799X-RAY DIFFRACTION99.9
4.43-5.070.181280.16762790X-RAY DIFFRACTION99.93
5.07-6.380.2651480.20642818X-RAY DIFFRACTION99.93
6.39-57.640.18441330.17742900X-RAY DIFFRACTION98.96
Refinement TLS params.Method: refined / Origin x: -29.1296860495 Å / Origin y: -5.8080576698 Å / Origin z: 38.7259386249 Å
111213212223313233
T0.44806135674 Å2-0.0935286948849 Å20.0177057378372 Å2-0.339612785784 Å20.00351652423645 Å2--0.426455600894 Å2
L0.718063574604 °2-0.0721275000377 °20.159476477709 °2--0.117694032392 °2-0.0378392875973 °2--0.315904165889 °2
S0.0794493413402 Å °-0.21492700014 Å °0.0103660972314 Å °0.0199972757267 Å °-0.0517228443206 Å °-0.0179970338666 Å °-0.0579468289817 Å °-0.0353776615169 Å °-0.0260584934792 Å °
Refinement TLS groupSelection details: all

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